Non-alloying core shell nanoparticles

ABSTRACT

The present invention relates composite core/shell nanoparticles and a two-step method for their preparation. The present invention further relates to biomolecule-core/shell nanoparticle conjugates and methods for their preparation. The invention also relates to methods of detection of biomolecules comprising the biomolecule or specific binding substance-core/shell nanoparticle conjugates.

CROSS-REFERENCE

[0001] This application claims the benefit of priority from U.S.Provisional application No. 60/293,861, filed May 25, 2001, which isincorporated by reference in its entirety. The work reported in thisapplication has been supported, in part, by NSF grant no. CHE-9871903;ARO grant no. DAAG55-97-1-0133, and AFOSR grant no. DURINT. Accordingly,the U.S. government may have some rights to the invention.

FIELD OF INVENTION

[0002] The present invention relates to core/shell nanoparticles,materials based on core/shell nanoparticles, kits containing core/shellnanoparticles, and methods of making and using core/shell nanoparticlesfor the detection of target molecules, including nucleic acids,peptides, and proteins. In particular, the present invention relates tospecific binding substance-modified core/shell nanoparticles such asDNA-modified core/shell nanoparticles and their use for detecting targetmolecules such as nucleic acids.

BACKGROUND OF INVENTION

[0003] In 1996, a method was reported for utilizing biomolecules, suchas DNA, and their molecular recognition properties to guide the assemblyof nanoparticle building blocks modified with complementary recognitionelements into functional materials.¹ These materials have found wideapplication in the development of highly sensitive and selectivediagnostic methods for DNA.² This material synthesis approach has beenextended to a wide range of biomolecules, including peptides andproteins,³ and a modest collection of nanoparticles including gold andsemiconductor quantum dots.⁴⁻⁹ In each case, when a new nanoparticlecomposition is designed, new modification methods must be developed forimmobilizing biomolecules on the surface of the particles of interest.This approach has been extensively utilized but with limited success.The methods for modifying gold nanoparticles have now been optimized andgeneralized for a wide range of particle sizes and surface compositions,including spheres and rods.^(1,2,4,10) Gold particles are particularlyeasy to modify because they are often stabilized with a weakly bindinglayer of charged ligands (e.g. citrate) that can be replaced withmolecules with chemical functionalities that bind more strongly (e.g.thiols, amines, and disulfides) to their surfaces than these ligands.The CdSe and CdS quantum dots have proven more difficult to modifybecause they have a surfactant layer that is very strongly bound totheir surfaces and, consequently, difficult to displace.⁵ No successfulroutes have been developed for creating stable oligonucleotideconjugates with silver nanoparticles, primarily because they tend tochemically degrade under conditions used to effect DNA hybrization. Amajor advance would be to devise a method for designing particles withthe physical properties of a chosen nanoparticle composition but thesurface chemistry of gold. Herein, a low temperature method is providedfor generating core/shell particles consisting of a silver core and anon-alloying gold shell that can be readily functionalized witholigonucleotides using the proven preparatory methods for pure goldparticle oligonucleotide conjugates.^(2d) Moreover, the novelnanoparticle composition can be used to access a colorimetric detectionsystem distinct from the pure gold system.^(2a,2d)

BRIEF SUMMARY OF THE INVENTION

[0004] The present invention relates to composite core/shellnanoparticles, compositions and kits including these core/shellnanoparticles, and methods for preparing and using composite core/shellnanoparticles, particularly Ag/gold core/shell nanoparticles, for thedetection of target molecules such as nucleic acids, proteins and thelike. These Ag/gold core/shell nanoparticles were prepared by reductionof HAuCl₄ by NaBH₄ in the presence of Ag-nanoparticle “templates” andcharacterized by UV-vis spectroscopy, transmission electron microscopy(TEM), and energy dispersive X-ray (EDX) microanalysis. Significantly,these particles do not alloy, yielding structures with the opticalproperties of silver and the surface chemistry and high stability of Au.Experimental and theoretical data support the structuralcharacterization of these novel materials as silver cores (˜12 nm indiameter) coated with approximately one atomic monolayer of gold(˜3 Å).The core/shell nanoparticles may be further modified withalkanethiol-oligonucleotides forming structures that undergo reversiblehybridization with complementary oligonucleotides to form extendednanoparticle network structures. By spotting aliquots of a solutioncontaining the oligonucleotide-modified nanoparticles without and withDNA target on a reverse-phase alumina plate, a distinct colorimetrictransition from yellow to dark brown can be observed by the naked eye.The optical properties of the dispersed and aggregated core/shellparticles form a new colorimetric channel for nanoparticle based DNAdetection.

[0005] Accordingly, one object of the invention is to providestraightforward method of preparing core/shell nanoparticles with theoptical, and many of the physical, properties of silver but thestability of gold. The surfaces of these nanoparticles can be modifiedwith a variety of moieties such as, for example, natural and syntheticpolymers, molecules capable of selective molecular recognitionincluding, but not limited to, nucleotides, nucleosides, poly- oroligonucleotides, proteins, peptides, carbohydrates, sugars, andhaptens, thereby providing useful biorecognition properties to thenanoparticles.

[0006] Another object of the invention is to provide a general methodfor preparing core/shell particles with tailorable physical propertiesby virtue of choice of core, e.g., Fe₃O₄, Cu or Pt, but the surfacechemistry and stability of the native, and oligonucleotide modified,pure gold particles.

[0007] Another object of the invention is to provide methods fordetection of molecules capable of selective molecular recognitioncomprising use of core/shell nanoparticle probes. These methods comprisecontacting the core/shell nanoparticle probes with one or a plurality oftarget molecules under conditions that allow for selective molecularrecognition, and the detection of an optical change. The physicalproperties of the particular core/shell nanoparticle probes can allowfor various additional steps in these methods such as, for example,inducing their migration through application of electrical or magneticfields.

[0008] Another object of the invention is to provide nanomaterials basedon the core/shell nanoparticles of the invention.

[0009] These and other objects of the invention will become apparent inlight of the detailed description below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 illustrates (A) a TEM image of Ag/Au core/shellnanoparticles; (B) EDX spectra of silver core nanoparticles (dottedline) and Ag/Au core/shell nanoparticles (solid line) wherein L and Msignify electron transitions into the L and M shell of the atoms,respectively, from higher states; (C) UV-vis spectra of silver core(dotted line) and Ag/gold core/shell (solid line) wherein the insetshows the calculated extinction spectra of silver nanoparticles (dottedline) and Ag/Au core/shell nanoparticles (solid line); (D) Thermaldenaturization curve of aggregates formed from hybridizedoligonucleotide modified Ag/Au core/shell nanoparticles in buffersolution (0.3 M NaCl and 10 mM phosphate buffer, pH=7). The inset showsthe UV-vis spectra of dispersed oligonucleotide-modified Ag/Aucore/shell nanoparticles (solid line) and aggregated (dotted line)oligonucleotide-modified Ag/Au core/shell nanoparticles formed viahybridization. The base sequences are given in FIG. 2A.

[0011]FIG. 2 illustrates (A) Mercaptoalkyl-oligonucleotide-modifiedAg/Au core/shell particles and an oligonucleotide target. Represents thecore/shell nanoparticle and “˜” represents a propyl (left) or hexyl(right) group linking S to the oligonucleotide probe. DNA spot testusing: (B) 12.4-nm Ag/gold nanoparticle probes and (C) 13-nm goldnanoparticle probes: (I) without target, (II) with target at roomtemperature, (III) with target at 58.0° C., a temperature above theT_(m) (53.0° C.) of the hybridized DNA.

[0012]FIG. 3 illustrates the UV-VIS spectra of a Pt core (dotted line)and Pt/gold core/shell nanoparticles (solid line).

[0013]FIG. 4 illustrates the UV-VIS spectra of gold growth on thesurface of Fe₃O₄ nanoparticles at 0, 0.3 nm, 0.6 nm, and 0.9 nmthickness.

[0014]FIG. 5 illustrates the behavior of Fe₃O₄/gold core/shell particlesas super paramagnetic particles in the presence of an applied magneticfield. In the presence of a magnetic field, a solution containing themagnetic gold nanoparticles appears red. When a magnetic force isapplied over a period of 2 hours, the solution becomes colorless as thenanoparticles migrate towards the magnetic force.

[0015]FIG. 6 illustrates the core/shell approach to magnetic goldnanaparticles.

[0016]FIG. 7 illustrates a comparison of the relative stabilities of Ag,Ag/Au alloy, and Ag@Au core/shell nanoparticle-DNA conjugates atdifferent salt concentrations.

DETAILED DESCRIPTION OF THE INVENTION

[0017] In one aspect the present invention provides for core/shellnanoparticles, comprising a nanoparticle core and a gold shell. The corematerial can comprise any nanoparticle known to those of skill in theart including, but not limited to, metal, semiconductor, and magneticnanoparticles. In a preferred embodiment, the core material is comprisedof metal or magnetic nanoparticles including, but not limited to, Ag,Pt, Fe, Co, Ni, FePt, FeAu, Fe₃O₄, and Co₃O₄. Methods for preparing suchnanoparticles are well known in the art. For example, see, e.g. Schmid,G. (ed.) Clusters and Colloids (VCH, Weinheim, 1994); Hayat, M. A. (ed.)Colloidal Gold: Principles, Methods, and Applications (Academic Press,San Diego, 1991); Massart, R., IEEE Taransactions On Magnetics, 17, 1247(1981); Ahmadi, T. S. et al., Science, 272, 1924 (1996); Henglein, A. etal., J. Phys. Chem., 99, 14129 (1995); Curtis, A. C., et al., Angew.Chem. Int. Ed. Engl., 27, 1530 (1988).

[0018] In yet another aspect the present invention provides a method forpreparation of non-alloying gold core/shell nanoparticles and productproduced therefrom. The method of the invention comprises providing aninner nanoparticle core, treating the core simultaneously with asolution comprising a gold salt and a solution comprising a reducingagent, and isolating the core/shell nanoparticles. The method providesfor the first time a non-alloying gold shell surrounding a nanoparticlecore. These non-alloying gold core/shell nanoparticles exhibitsurprising superior spectroscopic properties not found in conventionalgold core/shell nanoparticles and can be functionalized with moleculessuch as nucleic acids and receptors, to produce nanoparticle conjugatesthat can be used for targeting and detecting target analytes such asnucleic acids, antigens, proteins, carbohydrates and other substances.

[0019] In practicing the method of the invention, the method can beperformed at any temperature favorable in producing a non-alloying goldshell surrounding the core. Generally, the temperature depends on thechoice of reaction solvent used to generate the gold shell. Suitable,but non-limiting, examples of reaction solvents include water, aqueousbuffer solutions, oleic acid and trioctylphosphine oxide. In practicingthis invention, trisodium citrate solution is preferred.

[0020] In practicing the method of the invention, the temperaturegenerally ranges from about 0° C. to about 45° C. in water or aqueousreaction solutions. For organic solvents, the temperature generallyranges from about 130° C. to about 180° C. when oleic acid andtrioctylphosphine oxide are used.

[0021] The gold salt can comprise any suitable gold salt including, butnot limited to, HAuCl₄, NaAuCl⁴, KAuCl₄, or KAu(CN)₂. In practicing theinvention, the preferred gold salt is HAuCl₄.

[0022] The reducing agent can comprise any suitable reducing agentcapable of reducing the valency of the gold that comprises the gold saltsolution including, but not limited to, NaBH₄, ascorbic acid, NH₂OH andN₂H₄. In practicing the invention, the preferred reducing agent isNaBH₄.

[0023] In one aspect of the invention, the core/shell nanoparticles havespecific binding substances bound to the gold shell surrounding thenanoparticle. The specific binding substance may be natural andsynthetic nucleic acids, natural and synthetic polypeptides, antibodies,Fab and Fab′ antibody fragments, biotin, avidin and haptens such asdigoxin. Those skilled in these arts will recognize a wide variety ofspecific binding substances that can be linked to the gold shellsurrounding the nanoparticles.

[0024] In another aspect, the present invention provides for core/shellnanoparticle oligonucleotide conjugates, comprising a nanoparticle core,a gold shell surrounding the nanoparticle, and an oligonucleotideattached to the gold surface of the core/shell nanoparticle. Anysuitable method for attaching oligonucleotides onto a gold surface maybe used. A particularly preferred method for attaching oligonucleotidesonto a gold surface is based on an aging process described in U.S.application Ser. No. 09/344,667, filed Jun. 25, 1999; Ser. No.09/603,830, filed Jun. 26, 2000; Ser. No. 09/760,500, filed Jan. 12,2001; Ser. No. 09/820,279, filed Mar. 28, 2001; Ser. No. 09/927,777,filed Aug. 10, 2001; and in International application nos.PCT/US97/12783, filed Jul. 21, 1997; PCT/US00/17507, filed Jun. 26,2000; PCT/US01/01190, filed Jan. 12, 2001; PCT/US01/10071, filed Mar.28, 2001, the disclosures which are incorporated by reference in theirentirety. The aging process provides nanoparticle-oligonucleotideconjugates with unexpected enhanced stability and selectivity. Themethod comprises providing oligonucleotides preferably having covalentlybound thereto a moiety comprising a functional group which can bind tothe nanoparticles. The moieties and functional groups are those thatallow for binding (i.e., by chemisorption or covalent bonding) of theoligonucleotides to nanoparticles. For instance, oligonucleotides havingan alkanethiol, an alkanedisulfide or a cyclic disulfide covalentlybound to their 5′ or 3′ ends can be used to bind the oligonucleotides toa variety of nanoparticles, including gold nanoparticles.

[0025] The oligonucleotides are contacted with the nanoparticles inwater for a time sufficient to allow at least some of theoligonucleotides to bind to the nanoparticles by means of the functionalgroups. Such times can be determined empirically. For instance, it hasbeen found that a time of about 12-24 hours gives good results. Othersuitable conditions for binding of the oligonucleotides can also bedetermined empirically. For instance, a concentration of about 10-20 nMnanoparticles and incubation at room temperature gives good results.

[0026] Next, at least one salt is added to the water to form a saltsolution. The salt can be any suitable water-soluble salt. For instance,the salt may be sodium chloride, magnesium chloride, potassium chloride,ammonium chloride, sodium acetate, ammonium acetate, a combination oftwo or more of these salts, or one of these salts in phosphate buffer.Preferably, the salt is added as a concentrated solution, but it couldbe added as a solid. The salt can be added to the water all at one timeor the salt is added gradually over time. By “gradually over time” ismeant that the salt is added in at least two portions at intervalsspaced apart by a period of time. Suitable time intervals can bedetermined empirically.

[0027] The ionic strength of the salt solution must be sufficient toovercome at least partially the electrostatic repulsion of theoligonucleotides from each other and, either the electrostaticattraction of the negatively-charged oligonucleotides forpositively-charged nanoparticles, or the electrostatic repulsion of thenegatively-charged oligonucleotides from negatively-chargednanoparticles. Gradually reducing the electrostatic attraction andrepulsion by adding the salt gradually over time has been found to givethe highest surface density of oligonucleotides on the nanoparticles.Suitable ionic strengths can be determined empirically for each salt orcombination of salts. A final concentration of sodium chloride of fromabout 0.1 M to about 1.0 M in phosphate buffer, preferably with theconcentration of sodium chloride being increased gradually over time,has been found to give good results.

[0028] After adding the salt, the oligonucleotides and nanoparticles areincubated in the salt solution for an additional period of timesufficient to allow sufficient additional oligonucleotides to bind tothe nanoparticles to produce the stable nanoparticle-oligonucleotideconjugates. As will be described in detail below, an increased surfacedensity of the oligonucleotides on the nanoparticles has been found tostabilize the conjugates. The time of this incubation can be determinedempirically. A total incubation time of about 24-48, preferably 40hours, has been found to give good results (this is the total time ofincubation; as noted above, the salt concentration can be increasedgradually over this total time). This second period of incubation in thesalt solution is referred to herein as the “aging” step. Other suitableconditions for this “aging” step can also be determined empirically. Forinstance, incubation at room temperature and pH 7.0 gives good results.

[0029] The conjugates produced by use of the “aging” step have beenfound to be considerably more stable than those produced without the“aging” step. As noted above, this increased stability is due to theincreased density of the oligonucleotides on the surfaces of thenanoparticles which is achieved by the “aging” step. The surface densityachieved by the “aging” step will depend on the size and type ofnanoparticles and on the length, sequence and concentration of theoligonucleotides. A surface density adequate to make the nanoparticlesstable and the conditions necessary to obtain it for a desiredcombination of nanoparticles and oligonucleotides can be determinedempirically. Generally, a surface density of at least 10 picomoles/cm²will be adequate to provide stable nanoparticle-oligonucleotideconjugates. Preferably, the surface density is at least 15picomoles/cm². Since the ability of the oligonucleotides of theconjugates to hybridize with nucleic acid and oligonucleotide targetscan be diminished if the surface density is too great, the surfacedensity is preferably no greater than about 35-40 picomoles/cm².

[0030] As used herein, “stable” means that, for a period of at least sixmonths after the conjugates are made, the nanoparticles remaindispersed, a majority of the oligonucleotides remain attached to thenanoparticles, and the oligonucleotides are able to hybridize withnucleic acid and oligonucleotide targets under standard conditionsencountered in methods of detecting nucleic acid and methods ofnanofabrication.

[0031] In yet a further aspect the invention provides methods for thedetection of a target analytes such as nucleic acids comprisingcontacting the core/shell nanoparticle oligonucleotide conjugates of theinstant invention with a target nucleic acid sequence under conditionsthat allow hybridization between at least a portion of theoligonucleotides bound to the nanoparticle and at least a portion of thetarget nucleic acid sequence. In addition, protein receptors and otherspecific binding pair members can be functionalized witholigonucleotides and immobilized onto oligonucleotide-modifiednanoparticles to generate a new class of hybrid particles(nanoparticle-receptor conjugates) that exhibit the high stability ofthe oligonucleotide modified particles but with molecular recognitionproperties that are dictated by the protein receptor rather than DNA.Alternatively, one could functionalize a protein that has multiplereceptor binding sites with receptor-modified oligonucleotides so thatthe protein receptor complex could be used as one of the buildingblocks, in place of one of the inorganic nanoparticles, in the originalnanomaterials assembly scheme discussed above. The use of these novelnanoparticle-receptor conjugates in analyte detection strategies havebeen evaluated in a number of ways including identification of targetsand screening for protein-protein interactions. For suitablehybridization conditions for nucleic acid detection, and methods forpreparing nanoparticle-receptor conjugates are described in U.S.application Ser. No. 09/344,667, filed Jun. 25, 1999; Ser. No.09/603,830, filed Jun. 26, 2000; Ser. No. 09/760,500, filed Jan. 12,2001; Ser. No. 09/820,279, filed Mar. 28, 2001; Ser. No. 09/927,777,filed Aug. 10, 2001; and in International application nos.PCT/US97/12783, filed Jul. 21, 1997; PCT/US00/17507, filed Jun. 26,2000; PCT/US01/01190, filed Jan. 12, 2001; PCT/US01/10071, filed Mar.28, 2001, the disclosures which are incorporated by reference in theirentirety. Once a core/shell nanoparticle conjugate of the inventionbinds to a target molecule, a change in the optical characteristics ofthe core/shell nanoparticle conjugates can be readily detected. Inanother embodiment the detection step is performed in the presence of anapplied magnetic field which further enhances hybridization or bindingof the nanoparticle conjugate with the target molecule such as a nucleicacid.

[0032] The invention further provides a method of nanofabrication basedon the core-shell nanoparticle conjugates of the invention.Nanostructures and methods for prepare the materials from nanoparticleshave been described in U.S. application Ser. No. 09/344,667, filed Jun.25, 1999; Ser. No. 09/603,830, filed Jun. 26, 2000; Ser. No. 09/760,500,filed Jan. 12, 2001; Ser. No. 09/820,279, filed Mar. 28, 2001; Ser. No.09/927,777, filed Aug. 10, 2001; and in International application nos.PCT/US97/12783, filed Jul. 21, 1997; PCT/US00/17507, filed Jun. 26,2000; PCT/US01/01190, filed Jan. 12, 2001; PCT/US01/10071, filed Mar.28, 2001, the disclosures which are incorporated by reference in theirentirety. The method comprises providing at least one type of linkingoligonucleotide having a selected sequence, the sequence of each type oflinking oligonucleotide having at least two portions. The method furthercomprises providing one or more types of core/shell nanoparticles havingoligonucleotides attached thereto, the oligonucleotides on each type ofnanoparticles having a sequence complementary to a portion of thesequence of a linking oligonucleotide. The linking oligonucleotides andnanoparticles are contacted under conditions effective to allowhybridization of the oligonucleotides on the nanoparticles to thelinking oligonucleotides so that a desired nanomaterials ornanostructure is formed.

[0033] The invention provides another method of nanofabrication. Thismethod comprises providing at least two types of core-shellnanoparticles of the invention having oligonucleotides attached thereto.The oligonucleotides on the first type of nanoparticles have a sequencecomplementary to that of the oligonucleotides on the second type ofnanoparticles. The oligonucleotides on the second type of nanoparticleshave a sequence complementary to that of the oligonucleotides on thefirst type of nanoparticle-oligonucleotide conjugates. The first andsecond types of nanoparticles are contacted under conditions effectiveto allow hybridization of the oligonucleotides on the nanoparticles toeach other so that a desired nanomaterials or nanostructure is formed.

[0034] The invention further provides nanomaterials or nanostructurescomposed of core-shell nanoparticles having oligonucleotides attachedthereto, the nanoparticles being held together by oligonucleotideconnectors.

[0035] The following examples serve to illustrate certain embodiments ofthe present invention, and do not limit it in scope or spirit. Certainobvious alternatives and variations will be apparent to those of skillin the art.

EXAMPLE 1 Synthesis of Ag/Au Core/Shell Nanoparticles Prepared via aTwo-Step Synthesis

[0036] This Example illustrates the inventive process for preparingAg/Au core/shell nanoparticles. In part A, methods for preparingsilvercores are described. In part B, a method for preparing Ag/goldcore/shell nanoparticles is provided. Silver nanoparticles are desiredcompositions for building blocks in material synthesis and as biologicallabels for two important reasons: (1) silver particles exhibit a surfaceplasmon band between ˜390 and 420 nm, depending on the particle size;¹¹this is a spectral regime that is distinct from that of gold(520-580nm). (2) The extinction coefficient of the surface plasmon band for asilver particle is approximately 4 times as large as that for an goldparticle of the same size.¹² Therefore, silver particles functionalizedwith DNA would provide not only an opportunity to tailor the opticalproperties of DNA/nanoparticle composite structures but also routes tonew diagnostic systems that rely on the position and intensity of thesurface plasmon band (e.g. calorimetric systems based on absorption orscattering, or SPR and SERS detection systems).

[0037] Experimentally, it has been determined that silver nanoparticlescannot be effectively passivated by alkylthiol-modified-oligonucleotidesusing the established protocols for modifying goldparticles.² Indeed,silver particles prepared via such methods irreversibly aggregate whenheated in a solution with a salt concentration necessary to effect DNAhybridization (0.05 M NaCl or greater). Herein, a core/shell approachwas applied to overcome this problem. In this approach, a thin goldshellwas grown upon a silver nanoparticle, forming a particle with a goldouter surface that can be easily modified withalkylthiol-oligonucleotides. This approach could be generalized toprepare other particles such as Cu and Pt to create a series ofcore/shell particles with tailorable physical properties by virtue ofchoice of core but the surface chemistry and stability of the native,and oligonucleotide modified, pure gold particles.

[0038] A. Preparation of Silver Nanoparticle Cores

[0039] Silver nanoparticles were synthesized silver nanocrystals byreduction of silver nitrate by sodium borohydride in a trisodium citratesolution. Two methods for synthesizing the silver nanocrystals aredescribed below and the resulting core nanocrystals are compared.

[0040] Method No. 1: AgNO₃ (2.2 mg) and sodium citrate dihydrate (8.2mg) were dissolved in 99 ml of Nanopure water in a 250-ml flask. Withstirring and under Ar, this flask was placed in a ice bath for 15 min.Then 1 ml of sodium borohydride solution (0.14 M) was injected into thesolution. After stirring for 1 hr, the solution was warmed to roomtemperature. The silver nanoparticles (˜12 nm in diameter) wereobtained. Without further purification, these silver nanoparticles couldbe directly used for the gold shell growth.

[0041] Method No. 2: AgNO₃ (2.2 mg) and sodium citrate dihydrate (8.2mg) were dissolved in 98 ml of Nanopure water in a 250-ml flask. Withstirring and under an Ar atmosphere, this flask was placed in an icebath for 15 min. Then 1 ml of sodium borohydride solution (0.14 M) wasinjected into the solution. After stirring for 1 hr, the solution waswarmed to room temperature. The Ag nanoparticles (˜12 nm in diameter)were obtained. Bis(p-sulfonatophenyl)-phenylphosphine (BSPP, 17 mg) wasput into the silver nanoparticle solution and stirred overnight. Thesilver nanoparticles were subsequently purified and isolated by gradientcentrifugation between 12 kRPM˜20 kRPM. The resulting silvernanoparticle-containing aliquots from the precipitation were combined,and dispersed in Nanopure water.

[0042] Comparison results: Silver particles prepared by method no. 2have better size distribution compared with those prepared by method no.1 (σ=18% for method no. 2; σ=30% for method no. 1). Subsequent studieshave shown, however, that silver particles prepared by either methodserve well as cores for generating silver/gold core/shell nanoparticles.

[0043] B. Preparation of Silver/Gold Core/Shell Nanoparticles

[0044] This step describes gold shell growth on the surface of silvercores described above. For silver nanoparticles, gold shells were grownon the silver core surface by reduction of HAuCl₄ with the reducingsilverent NaBH₄. The reduced gold has affinity for the silver surface,in part, because of the low surface chemical potential of the silvernanoparticles and near-zero lattice mismatch between these twomaterials. Two methods for growing gold shells on silver corenanocrystals are described below and the resulting core/shellnanoparticles were compared. silver core particles were prepared bymethod no. 1 described above.

[0045] Method No. 1: Gold shells (approximately one-monolayer thick)were grown on the surface of the silver nanoparticles (0.25 nmol ofsilver particles in 100 ml of 0.3 mM sodium citrate aqueous solution) bysimultaneous dropwise addition, at a rate of between about 50 μL˜600μL/min., of HAuCl₄ and NaBH₄ solutions (in Nanopure water) at 0° C. tothe silver nanoparticle suspension. The simultaneous dropwise additionof dilute gold precursors inhibits the formation of gold clusternucleation sites by keeping the concentration of these gold formingreagents at about 2 μM. After enough HAuCl₄ and NaBH₄ was added to thenanoparticles to produce one monolayer of gold on the particles (seeEquation 1 for a calculation of shell thickness), addition was halted.

V _(core)=4/3*π*R ³;

V _(core/shell)=4/3*π*(R+a)³,  Equation 1:

[0046] wherein a is the shell thickness, (0.3 nm for 1 monolayer of Au);

V _(shell) =V _(core/shell) −V _(core);

N _(shell) =d _(shell) *V _(shell) /FW _(shell);

[0047] wherein,V_(shell) is volume of shell;

[0048] N_(shell) is the amount in mole of the shell;

[0049] d_(shell) is density of shell materials, (for gold, d=19.3 g/ml);

[0050] FW_(shell,) the formula weight of shell materials, (for gold,FW=196.97 amu)

[0051] Gold was added 5% excess, calculated assuming 12-nm spheres: 0.8mg of HAuCl₄.3H₂O and 3.7 mg of NaBH₄. Once 5% excess was achieved,addition of the solutions was stopped (halting formation of the shell)and 30 μmol of Bis(p-sulfonatophenyl)phenylphosphine (BSPP) was added.The silver/gold core/shell nanoparticles were then purified bycentrifugation and dispersed in Nanopure water (12.4 nm in diameter,(σ=18%)), giving a 96% yield and a ratio of silver to gold of about5.5:1.

[0052] Method No. 2: Gold shells (approximately one-monolayer thick)were grown on the surface of the silver nanoparticles (0.25 nmol ofsilver particles in 100 ml of 0.3 mM sodium citrate aqueous solution) bysimultaneously treating them with HAuCl₄ (2 mM) and NaBH₄ (6 mM) viadropwise addition at room temperature at a rate of between about 50μL˜600 μL/min. The simultaneous dropwise addition of dilute goldprecursors inhibits the formation of gold cluster nucleation sites bykeeping the concentration of these gold forming reagents at about 2 μM.After sufficient HAuCl₄ and NaBH₄ were added to the nanoparticles toproduce one monolayer of gold on the particles (5% excess, calculatedassuming 12-nm spheres: 0.8 mg of HAuCl₄.3H₂O and 3.7 mg of NaBH₄), thereaction was stopped and 30 μmol of BSPP was added. The silver/goldcore/shell nanoparticles were then purified by centrifugation anddispersed in nanopure water, giving a weight percent yield of about 90%,and average particle size of about 12.5 nm, and an silver to gold ratioof about 6.3:1.

[0053] Comparison results: The core/shell nanoparticles produced viamethod no. 1 (synthesis at 0° C.) were found to have better stability in0.5 M NaCl solution compared to core/shell nanoparticles produced bymethod no. 2 (synthesis at room temperature). This result may be due, inpart, to a slower rate of shell growth at 0° C. than the growth rate atroom temperature.

[0054] (c) Discussion

[0055] Silver nanoparticles were prepared by literature methods.¹³ Theparticles were then passivated with BSPP (0.3 mM), purified by gradientcentrifugation (collecting the primary fraction; ˜12 nm in diameter),and dispersed in Nanopure water. Gold shells, approximatelyone-monolayer thick, were grown on the surface of the silvernanoparticles (0.32 nmol of silver particles in 100 mL of 0.3 mM sodiumcitrate aqueous solution) by simultaneously treating them with HAuCl₄and sodium borohydride via dropwise addition at 0° C. The reduced goldhas an affinity for the silver surface, in part, because of its nearzero lattice mismatch.¹⁴ The simultaneous dropwise addition of dilutegold precursors inhibits the formation of gold cluster nucleation sitesby keeping the concentration of these gold forming reagents at about 2μM. After enough HAuCl₄ and NaBH₄ were added to the nanoparticles toproduce one monolayer of gold on the particles (5% excess, calculatedassuming 12-nm spheres: 0.8 mg of HAuCl₄.3H₂O and 3.7 mg of NaBH₄), thereaction was stopped and 30 mM of BSPP was added. Then, the silver/goldcore/shell nanoparticles were purified by centrifugation and dispersedin nanopure water (12.4 nm in diameter particles, (σ=18%). FIG. 1A showsa TEM image of silver/gold core/shell nanoparticles which was obtainedusing a Hitachi 8100 electron microscopy. A typical TEM sample wasprepared by depositing one drop of nanoparticles solution onto a carboncoated copper grid. The excess solution was wicked away by filter paperand dry in vacuum. The silver:gold ratio in these core/shell particleswas determined to be 5.2:1 by energy dispersive X-ray (EDX)microanalysis of the particles (FIG. 1B). FIG. 1B illustrates an EDXspectrum of silver core particles (dotted line) and silver/goldcore/shell particles (solid line). L and M signify electron transitionsinto the L and M shell of the atoms, respectively, from higher states.EDX analysis was performed on a field emission scanning-electronmicroscopy (FESEM) Hitachi 4500. The SEM samples were prepared bydepositing of one drop of nanoparticle solution on a silicon plate. Thesilver:gold ratio corresponds to an gold shell thickness of 3.1+/−0.6 Å,which correlates with approximately one monolayer of gold atoms.

[0056] Significantly, the extinction spectrum of the core/shellparticles is very similar to that for the citrate-stabilized pure silverparticles. The surface plasmon band of the silver remains at the samewavelength but is dampened by about 10%, and the gold plasmon band isobserved as a slight buckle at 500 nm. These spectral features providestrong evidence for gold shell growth. It should be noted that usingdifferent procedures, others have prepared gold-coated silvernanoparticles.¹⁵ However, those procedures lead to silver/goldalloys;^(15a) the extinction spectra of such particles exhibitcharacteristic red shifting and broadening of the plasmon resonance.Moreover, if one intentionally makes a solution of alloyed silver/goldparticles, they can be easily distinguished from core/shell particleswith comparable silver/gold ratios (see Supporting Information). Indeed,the core/shell silver/gold nanoparticles prepared by the methods of theinstant invention retain the optical properties of the core with noobserved red shifting of the silver plasmon band, (FIG. 1C). Using Mietheory, the extinction spectrum of a particle consisting of an 11.8 nmsilver core and a monolayer gold shell was calculated.¹¹ The calculatedspectrum was almost superimposable with the experimentally measuredspectrum of the particles, (FIG. 1C, inset). FIG. 1C illustrates theUV-visible spectra of silver core (dotted line) and silver/goldcore/shell (solid line) wherein the inset shows the calculatedextinction spectra of silver particles (dotted line) and silver/goldcore/shell particles (solid line). The UV/Vis spectra were obtainedusing a HP 8453 diode array spectrophotometer.

EXAMPLE 2 Preparation of Silver/Gold Core/ShellNanoparticle-Oligonucleotide Conjugates

[0057] This Example describes the preparation of silver/gold core/shellnanoparticle oligonucleotide conjugates as probes for detecting a targetnucleic acid. Two methods were employed and the resulting probes werethen compared for stability. The oligonucleotide sequences used inmaking the conjugates are shown in FIG. 2a. These sequences weresynthesized using standard phosphoramidite chemistry according to theliterature. (James J. Storhoff, Robert Elghanian, Robert C. Mucic, ChadA. Mirkin, and Robert L. Letsinger, J. Am. Chem. Soc., 1998, 120, 1959).

[0058] (a) Preparation of Core/Shell Nanoparticle Conjugates

[0059] Method No. 1: Nanoparticle probes with appropriate probeoligonucleotides were prepared by derivatizing 10 mL of aqueouscore/shell nanoparticle colloid (from method no. 1) with 8˜10 OD (inabout 500 uL) of alkanethiol-oligonucleotide (final oligonucleotideconcentration is about 2 μM). After standing overnight (about 15 h), thesolution was brought to 10 mM phosphate buffer (pH 7), using 100 mMconcentrated phosphate stock buffer, and salt (from a 2 M aqueous NaClsolution) added to 0.05 M NaCl after 0.5 h, allowed to stand for about 8h, then further addition of NaCl to 0.1 M, and after another standingtime of about 8 h, another addition of NaCl to about 0.3 M and allowedto stand for a final ˜8 h. To remove excess DNA, colloids werecentrifuged for 30 min at 18,000 rpm using 1.5 mL eppendorf tubes.Following removal of the supernatant, the oily precipitate was washedwith a volume equal to the discarded supernatant with 0.3 M NaCl, 10 mMphosphate buffer (pH 7) solution, centrifuged, and dispersed in 0.3 MNaCl, 10 mM phosphate buffer (pH 7), 0.01% azide solution. The finalcolloids were refrigerated and stored for later use.

[0060] Method No. 2: Nanoparticle probes with appropriate probeoligonucleotides were prepared by derivatizing 10 mL of aqueous colloidwith 8˜10 OD of alkanethiol-oligonucleotide (final oligonucleotideconcentration is about 2 μM). After standing overnight (˜15 h), thesolution was brought to 10 mM phosphate buffer (pH 7), using 100 mMconcentrated phosphate stock buffer, and salt added to 0.1 M NaCl,allowed to stand for about 20 h, and again, salt added to 0.3 M afteranother ˜8 h. The mixture was allowed to stand for about 4 to 8 hours.To remove excess DNA, colloids were centrifuged for 30 min at 18,000 rpmusing 1.5 mL eppendorf tubes. Following removal of the supernatant, theoily precipitate is washed with 0.3 M NaCl, 10 mM phosphate buffer (pH7) solution in the same volume as the discarded supernatant,centrifuged, and dispersed in 0.3 M NaCl, 10 mM phosphate buffer (pH 7),0.01% azide solution. The final colloids were refrigerated and storedfor later use.

[0061] (b) Evaluation of Stability of Core/Shell NanoparticleOligonucleotide Conjugates

[0062] The core/shell nanoparticle oligonucleotide conjugates preparedby the two methods described above were compared using a saltingprocedure as described in each of the above 2 methods.

[0063] By method 1, the salt concentration was increased from 0.05 MNaCl to 0.1 M NaCl, and then to 0.3 M NaCl. By method 2, the saltconcentration was increased in two steps: directly to 0.1 M NaCl andthen to 0.3 M NaCl. Method 1 generates a higher qualitynanoparticle-oligonucleotide conjugate when compared with those preparedby method 2. Via method 2, about 15% of the nanoparticle-oligonucleotideconjugates are not of adequate quality. Core/shellnanoparticle-oligonucleotide conjugate quality is evaluated by UV-Visspectroscopy. Acceptable quality conjugates show a UV-Vis spectrum withthe surface plasmon absorption peak centering at 400 nm, while poor(inadequate) quality conjugates show an absorption peak which issignificantly damped and red-shifts to 450-550 nm.

[0064] (c) Discussion

[0065] The surface modification of these core/shell nanoparticles with3′- and 5′-alkanethiol-capped oligonucleotides was accomplished using aprocedure identical to the one used for 13-nm gold particles.^(2d)Significantly, the oligonucleotide-modified core/shell particles exhibitthe stability of oligonucleotide modified particles prepared using puregold nanoparticles and can be suspended in 1M NaCl solutionindefinitely. This represents a significant advantage over theoligonucleotide modified silver/gold alloy particles which irreversiblyaggregate under comparable solution conditions and do not exhibit thestability of the oligonucleotide-modified core/shell particles of theinstant invention.

[0066] Moreover, the core/shell particles undergo hybridization withcomplementary linking oligonucleotides to form aggregated structureswith a concomitant darkening of the solution; (FIG. 2). Like theoligonucleotide modified pure gold nanoparticles, the nanoparticlescomprising these silver/gold core/shell aggregate structures can bedisassembled by heating the aggregates above the “melting temperature”(T_(m)) of the duplex linkers (FIG. 1D). UV-vis spectroscopy shows ared-shifting and dampening of the plasmon resonance of the core/shellparticles upon DNA-induced assembly, (FIG. 1D, inset). FIG. 1Dillustrates the thermal denaturation (“melting”) curve of aggregatesformed from hybridized oligonucleotide modified silver/gold core/shellparticles in buffer solution (0.3 M NaCl and 10 mM phosphate buffer,pH=7). The oligonucleotide sequences are provided in FIG. 2A. The FIG.1D inset shows the UV-visible spectra of dispersedoligonucleotide-modified silver/gold core/shell particles (solid line)and aggregated (dotted line) oligonucleotide-modified silver/goldcore/shell particles formed via hybridization. UV-Vis spectra of silverand silver/gold core/shell particles (FIG. 1C and inset of FIG. 1D) wereobtained using a HP 8453 diode array spectrophotometer. The thermaldenaturation experiment (FIG. 1D) was performed using an HP 8453 diodearray spectrophotometer equipped with a HP 89090a Peltier temperaturecontroller. The UV-Vis signature of the silver/gold core/shellprobe/target oligonucleotide aggregates was recorded at 1 min intervals,as the temperature was increased from 25 to 70° C. with a holding timeof 1 min/deg.

[0067] The particle assembly process induced by the complementary DNAalso can be monitored on a C₁₈-reverse-phase alumina TLC plate, allowingfor comparison with the pure gold system. The spot test results shown inFIG. 2b and 2 c were obtained as follows: a solution of the appropriateoligonucleotide target (24 pmol, 3 μL) was added to a 600 μL thin-wallPCR tube containing 200 μL of each silver/gold core/shellnanoparticle-oligonucleotide conjugates. After standing for 30 min atroom temperature, the solution was transferred to a temperaturecontrolled electro thermal heater. After the set-point temperature wasreached (monitored with an ethanol thermometer, 0.5° C. increments), themixture was allowed to equilibrate for 5 min at which time 2.5 μLaliquots of the silver-gold probe/target oligonucleotide solution weretransferred with a pipet onto the reverse-phase alumina plate andallowed to dry.

[0068] As shown in FIG. 2, with the core shell particles, a distinctyellow-to-dark brown color change is observed upon particle assembly inthe presence of complementary target, FIGS. 2B-I and 2B-II. Note thatwhen the solution temperature is above the T_(m) of the DNA duplexlinkers, a yellow spot is formed on the reverse phase alumina support,FIG. 2B-III. When one compares the properties of these new silver/goldcore/shell probes with those derived from pure gold nanoparticles (withidentical oligonucleotide sequences), FIG. 2C, one realizes that thecore/shell particles provide a route to a second colorimetric changedistinct from the gold system that ultimately could be used formonitoring two different oligonucleotide targets in one sample. Suchcapabilities could be important for both research-based and clinicalgenomic assays where multicolor formats are essential.¹⁶

EXAMPLE 3 Comparison of Silver, Silver/Gold Core/Shell and Silver/GoldAlloy Nanoparticle Oligonucleotide Conjugates

[0069] In this Example, the silver/gold core/shell nanoparticlesprepared as described in Example 1 (method no. 1) were compared to goldnanoparticles² and to silver/gold alloy nanoparticles.

[0070] The silver/gold alloy nanoparticles were prepared by the methodof Wang, Z. L.; El-Sayed, M. A. J. Phys. Chem.B 1999, 103, 3529.Following literature procedure, 0.8 mg of HAuCl₄.3H₂O and 1.8 mg ofsilverNO₃ were dissolved in 95 ml of nanopure water. The solution washeated to reflux, and 5 ml of 1% sodium citrate was added to thesolution. After refluxing an additional 30 min., the solution wasallowed to cool to room temperature.

[0071] The UV-Vis spectrum of the alloy particles exhibits a surfaceplasmon band at 428 nm with a full width at half-maximum (FWHM) of 90 nm(0.62 eV). In contrast, the UV-Vis spectrum of the silver/goldcore/shell nanoparticle, with a comparable silver/gold ratio, exhibits asurface plasmon band at 400 nm with a FWHM of 58 nm (0.45 eV). FIG. 7shows a comparison of the relative stabilities of Ag, Ag/Au alloy, andAg/Aucoreshell nanoparticle-DNA conjugates at different saltconcentrations. The surface plasmon bands were monitored at 400 nm forAg and Ag/Au particles, and at 430 nm for Ag/Au alloy particles,respectively.

[0072] The surface modification of these core/shell and alloynanoparticles with 3′- and 5′-alkanethiol-capped oligonucleotides wasaccomplished using a procedure identical to the one used for 13-nm goldparticles. See Storhoff, J. J.; Elghanian, R.; Mucic, R. C.; Mirkin, C.A.; Letsinger, R. L. J. Am. Chem. Soc. 1998 120, 1959. Significantly,the oligonucleotide-modified core/shell nanoparticles exhibit thestability of the pure gold nanoparticles and can be suspended in 1M NaClsolutions indefinitely. In contrast, the oligonucleotide-modifiedsilver/gold alloy particles irreversibly aggregate when brought to asalt concentration of 0.1 M.

[0073] Another way to evaluate stability of the particle/DNA conjugateuses a DNA melting test. The core/shell nanoparticle/DNA conjugate canreversibly hybridize with target DNA in a salt concentration range from0.1 to 1.0 M, and the resulting nanoparticle aggregates can “melt” offwhen heated above the melting temperature. Thishybridization/dehybridization process is completely reversible forcore/shell particles. The core/shell particle/DNA conjugates show nodegradation after 100 cycles. In sharp contrast, the silver/gold-alloyparticle/DNA conjugates irreversibly aggregate even under minimal saltconcentrations (˜0.05 M NaCl) necessary to effect hybridization ofoligonucleotides.

EXAMPLE 4 Preparation of Pt/Gold Core/Shell Nanoparticles

[0074] This Example describes the preparation of Pt/gold core/shellnanoparticles by the inventive process. In Part A, Pt core nanoparticleswere prepared by hydrogen reduction of K₂PtCl₄ in an overnight reaction.In Part B, goldshells were grown on the Pt cores.

[0075] (a) Preparation of Pt Core Nanoparticles

[0076] In a 500-ml three-neck flask, K₂PtCl₄ (8.3 mg) and sodiumpolyacrylate (20 mg) were dissolved in 200 ml of Nanopure water. H₂ wasbubbled into the reaction solution overnight with stirring. Thisresulted in Pt nanoparticles that were purified and isolated, yieldingnanoparticles of about 12 nm in diameter.

[0077] (b) Preparation of Pt/Gold Core/Shell Nanoparticles

[0078] 100 ml of 12-nm Pt nanoparticle solution (as prepared accordingto the above procedure) was put into a 250-ml three-neck flask. To thenanoparticle solution were added HAuCl₄ and NaBH₄ dropwise,simultaneously, at 0° C. The simultaneous dropwise addition of dilutegoldprecursors inhibits the formation of gold cluster nucleation sitesby keeping the concentration of these gold forming reagents at about 2μM. After sufficient amounts of HAuCl₄ and NaBH₄ were added to thenanoparticles to produce one monolayer of gold on the Pt nanoparticles(5% excess, calculated assuming 12-nm spheres: 16 mg of HAuCl₄.3H₂O and8 mg of NaBH₄), addition of these reagents to the reaction was stopped.UV-Vis spectra of Pt core and Pt/gold core/shell nanoparticle are shownin FIG. 3.

EXAMPLE 5 Preparation of Magnetic Fe₃O₄/Gold Core/Shell Nanoparticles

[0079] This Example describes the preparation of magnetic goldnanoparticles by the inventive process. In Part A, Fe₃O₄ magnetic corenanoparticles were prepared. In Part B, goldshells were grown on themagnetic cores. Other magnetic cores could be used in place of Fe₃O₄such as Co, Fe, Ni, FePt, and FeAu. FIG. 6 illustrates the core/shellapproach to preparing magnetic gold nanoparticles.

[0080] (a) Preparation of Fe₃O₄ Core Nanoparticles

[0081] In a typical synthesis, Fe₃O₄ nanoparticles were prepared asfollows. First, 0.86 g FeCl₂.4H₂O and 2.35 g FeCl₃.6H₂O were dissolvedin 50 mL nanopure water under an inert Ar_((g)) atmosphere. The solutionwas heated to 80° C. with vigorous stirring. A solution of 100 mg ofneat decanoic acid in 5 mL of acetone was added to the Fe solution,followed by 5 mL of 28% (w/w) NH₃/H₂O. Additional neat decanoic acid wasadded to the suspension in 5×0.2 g amounts over 5 min. The reaction wasallowed to proceed for 30 min at 80° C. with stirring to produce astable, water-based suspension. Following formation of the suspension,the reaction was cooled slowly to room temperature. The resulting Fe₃O₄nanoparticles (5.0 nmol) were treated further with Na₂S (8.0 mg)solution overnight to allow for sulfur exchange at the particle surface.Sulfur ions replace oxygen on the surface of the Fe₃O₄ nanoparticles,providing the growth site for the goldshell. This sulfur exchangeprocess is also necessary for the preparation of Co₃O₄ magnetic cores.

[0082] (b) Preparation of Fe₃O₄/Gold Core/Shell Nanoparticles

[0083] The procedure for growing goldshell is similar to that ofcore/shell silver/goldpreparation described in Example 1. The UV-Visspectrum, of the Fe₃O₄/gold shell growth is shown in FIG. 4. FIG. 5illustrates that in an applied magnetic field, Fe₃O₄/gold core/shellparticles behave as super paramagnetic particles. FIG. 5 illustratesthat the gold nanoparticles become colorless after 12 hours in amagnetic field.

EXAMPLE 6 Preparation of Magnetic Co/Gold Core/Shell Nanoparticles

[0084] This Example describes the preparation of magnetic goldnanoparticles by the inventive process. In Part A, Co magnetic corenanoparticles were prepared. In Part B, goldshells were grown on the Comagnetic cores.

[0085] (a) Preparation of Co Nanoparticle Cores

[0086] O-dichlorobenzene (15.9 g), trioctylphosphine oxide (0.1 g), and0.2 ml of oleic acid were placed into a 50-ml tri-neck flask, and heatedto 180° C. A solution of Co₂(CO)₈ (0.65 g in 3 ml of O-dichlorobenzene)was added by injection into the heated solution. After this addition,the reaction temperature was maintained at 180° C. for an hour. Thereaction solution was then cooled to room temperature. Co nanoparticlesof about 12 nm in diameter were produced in a yield of 95%.

[0087] (b) Preparation of Co/Gold Core/Shell Nanoparticles

[0088] The following is a typical coating protocol for Co/goldcore/shell nanoparticles.

[0089] After Co nanoparticles (0.01 μmol) were dissolved inO-dichlorobenzene (12 g) in a 50-ml tri-neck flask, trioctylphosphineoxide (0.1 g) was added in the Co solution. The solution was heated to180° C., at which point the gold-shell stock solutions 1 and 2 (seebelow) were added dropwise, simultaneously, to the hot reactionsolution, at a rate of about 50 μl-500 μl/min. After sufficient amountof stock solutions 1 and 2 were added (about 5% excess), the reactionsolution was maintained at 180° C. for another 30 mins. Subsequently,the reaction was cooled to room temperature in order to halt it.

[0090] The gold shell stock solutions were prepared as follows: stocksolution 1, HAuCl₄.3H₂O (0.1 g) and n-hexadecyltrimetyl ammonium bromide(0.1 g) were dissolved in O-dichlorobenzene (10 g); stock solution 2,1,1-hexadecanediol (0.12 g) was dissolved in O-dichlorobenzene (10 g).

[0091] The above examples merely serve to illustrate certain embodimentsof the present invention and do not serve to limit it in its scope orspirit.

REFERENCES

[0092] 1. Mirkin, C. A.; Letsinger, R. L., Mucic, R. C.; Storhoff, J. J.Nature 1996, 382, 607.

[0093] 2. (a) Elghanian, R.; Stohoff, J. J.; Mucic, R. C.; Letsinger, R.L.; Mirkin, C. A. Science 1997, 277, 1078. (b) Taton, T. A.; Letsinger,R. L.; Mirkin, C. A. Science 1999, 289, 1757. (c) Taton, T. A.; Lu, G.;Mirkin, C. A. J. Am. Chem. Soc. 2001, 123, 5164. (d) Storhoff, J. J.;Elghanian, R.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. J. Am.Chem. Soc. 1998 120, 1959.

[0094] 3. (a) Mann, S.; Shenton, W.; Li, M.; Connolly, S. Fitzmaurice,D. Adv. Mater. 2000, 12, 147. (b) Niemeyer, C. M.; Burger, W.; Peplies,J. Angew. Chem., Int. Ed. 2000, 37, 2265.

[0095] 4. Alivisatos, A. P.; Johnsson, K. P.; Peng, X.; Wilson, T. E.;Loweth C. J.; Bruchez, M. P., Jr.; Schultz, P. G. Nature 1996, 382, 609.

[0096] 5. Mitchell, G. P.; Mirkin, C. A.; Letsinger R. L. J. Am. Chem.Soc. 1999, 121, 8122.

[0097] 6. Chan, W. C. W.; Nie, S. Science 1998, 281, 2016.

[0098] 7. Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.Sundar V. C.; Mikulec F. V., Bawendi, M. G. J. Am. Chem. Soc. 2000, 122,12142.

[0099] 8. He, L.; Musick, D. M.; Nicewamer, S. R.; Ssalinas, F. G.;Benkovic, S. J. Natan, M. J.; Keating, C. D. J. Am. Chem. Soc. 2000,122, 9071.

[0100] 9. Pathak, S.; Choi, S. K.; Arnheim, N.; Thompson, M. E. J. Am.Chem. Soc. 2001, 123, 4103.

[0101] 10. Martin, B. R.; Derinody, D. J.; Reiss, B. D.; Fang, M.; Lyon,L. A.; Natan, M. J.; Mallouk, T. E. Adv. Mater. 1999, 11, 1021.

[0102] 11. Mulvaney, P. Langmuir 1996, 12, 788.

[0103] 12. Link, S.; Wang, Z. L.; El-Sayed, M. A. J. Phys. Chem.B1999,103, 3529.

[0104] 13. Ung, T.; Liz-Marzan, L. M.; Mulvaney, P. Langmuir 1998, 14,3740.

[0105] 14. Lide, D. R. Eds, Handbook of Chemistry and Physics, CRCpress: Boca Raton, 1992.

[0106] 15. (a) Mulvaney, P.; Giersig, M.; Henglein, A. J. Phys. Chem.1993, 97, 7061. (b) Rivas, L.; Sanchez-Cortes, S.; Garcia-Ramos; J. V.;Morcillo, G. Langmuir 2000, 16, 9722. (c) Ygeurabide U.S. Pat. No.6,214,560.

[0107] 16. Schrock. E.; duManoir, S.; Veldman, T.; Schoell. B.;Wienberg, J.; FergusonSmith, M. A.; Ning, Y.; Ledbetter, D. H.; BarAm,I.; Soenksen, D.; Garini, Y.; Ried, T. Science, 1996, 273, 494.

[0108] 17. C. Abdelghani-Jacquin et al., Langmair 2001, 17, 2129-2136.

1 3 1 35 DNA Artificial Description of artificial sequence Nanoparticleprobe 1 taacaataat ccctcaaaaa aaaaaaaaaa aaaaa 35 2 35 DNA ArtificialDescription of artificial sequence Nanoparticle probe 2 aaaaaaaaaaaaaaaaaaaa atccttatca atatt 35 3 30 DNA Artificial Description ofartificial sequence target sequence 3 gagggattat tgttaaatat tgataaggat30

We claim:
 1. An core/shell nanoparticle comprising: (a) an innermetal-containing nanoparticle core; and (b) an outer non-alloying goldshell surrounding the nanoparticle core.
 2. A core/shell nanoparticlespecific binding substance conjugate comprising: (a) an innermetal-containing nanoparticle core; (b) an outer non-alloying gold shellsurrounding the nanoparticle core; and (c) specific binding substanceattached to the gold shell.
 3. A core/shell nanoparticle oligonucleotideconjugate comprising: (a) an inner metal-containing nanoparticle core;(b) an outer non-alloying gold shell surrounding the nanoparticle core;and (c) oligonucleotides attached to the gold shell.
 4. The core/shellnanoparticle of claim 3 wherein the oligonucleotides have a sequencecomplementary to a portion of a sequence of a target nucleic acid. 5.The core/shell nanoparticle of claims 1, 2 or 3 wherein the innermetal-containing nanoparticle core comprises silver, Pt, Fe, Co, or Ni.6. The core/shell nanoparticle of claims 1, 2 or 3 wherein the innermetallic nanoparticle core comprises an alloy metal comprising FePt orFeAu.
 7. The core/shell nanoparticle of claims 1, 2 or 3 wherein theinner metal-containing nanoparticle core comprises a metal oxide.
 8. Thecore/shell nanoparticle of claims 1, 2 or 3 wherein the innermetal-containing nanoparticle core is magnetic.
 9. The core/shellnanoparticle of claim 7 wherein the inner metal-containing nanoparticlecore comprises Fe₃O₄ or Co₃O₄.
 10. The core/shell nanoparticle of claims1, 2 or 3 wherein the gold shell ranges from about 0.5 to about 2monolayers in thickness.
 11. The core/shell nanoparticle of claim 3wherein the oligonucleotides are attached to the nanoparticles in astepwise ageing process comprising (i) contacting the oligonucleotideswith the nanoparticles in a first aqueous solution for a period of timesufficient to allow some of the oligonucleotides to bind to thenanoparticles; (ii) adding at least one salt to the aqueous solution tocreate a second aqueous solution; and (iii) contacting theoligonucleotides and nanoparticles in the second aqueous solution for anadditional period of time to enable additional oligonucleotides to bindto the nanoparticles;
 12. The method of claim 11 wherein theoligonucleotides include a moiety comprising a functional group whichcan bind to a nanoparticle.
 13. The method of claim 11 wherein all ofthe salt is added to the water in a single addition.
 14. The method ofclaim 11 wherein the salt is added gradually over time.
 15. The methodof claim 11 wherein the salt is selected from the group consisting ofsodium chloride, magnesium chloride, potassium chloride, ammoniumchloride, sodium acetate, ammonium acetate, a combination of two or moreof these salts, one of these salts in a phosphate buffer, and acombination of two or more these salts in a phosphate buffer.
 16. Themethod of claim 15 wherein the salt is sodium chloride in a phosphatebuffer.
 17. The method of claim 11 wherein nanoparticle-oligonucleotideconjugates are produced which have the oligonucleotides present onsurface of the nanoparticles at a surface density of at least 10picomoles/cm².
 18. The method of claim 17 wherein the oligonucleotidesare present on surface of the nanoparticles at a surface density of atleast 15 picomoles/cm².
 19. The method of claim 18 wherein theoligonucleotides are present on surface of the nanoparticles at asurface density of from about 15 picomoles/cm² to about 40picomoles/cm².
 20. A nanostructure comprising the core/shellnanoparticles of claims 1 or
 2. 21. A silver/gold core/shellnanoparticle comprising: (a) an inner silver nanoparticle core; and (b)an outer non-alloying gold shell surrounding the nanoparticle core. 22.A magnetic core/shell nanoparticle comprising: (a) an inner Fe3O4nanoparticle core; and (b) an outer non-alloying gold shell surroundingthe nanoparticle core.
 23. The core/shell nanoparticle oligonucleotideconjugate of any of claims 2 or 3 exhibiting a surface plasmonabsorption peak at about 500 to 530 nm.
 24. A method for preparingcore/shell nanoparticles comprising the steps of: (a) providing innermetal-containing nanoparticle cores; (b) treating the innermetal-containing nanoparticle cores simultaneously with a solutioncomprising a gold salt and a solution comprising a reducing silverent at0° C. to produce a non-alloying gold shell surrounding the nanoparticlecores; and (c) isolating the core/shell nanoparticles.
 25. The methodaccording to claim 24 wherein the gold salt comprises HAuCl₄, NaAuCl₄,KAuCl₄, or KAu(CN)₂.
 26. The method according to claim 24 wherein thegold salt comprises HAuCl₄.
 27. The method according to claim 24 whereinthe reducing silverent comprises NaBH₄ or ascorbic acid.
 28. The methodaccording to claim 27 wherein the reducing silverent comprises NaBH₄.29. The method according to claim 24 wherein the gold salt and reducingsilverent are present at a ratio ranging from about 1:2 to about 1:20.30. A product produced by the method of
 24. 31. The product of claim 30wherein the gold shell thickness is about 0.15 to 0.6 nm.
 32. A methodof detecting nucleic acid bound to a surface comprising: (a) contactingthe surface with a solution comprising core/shell nanoparticleoligonucleotide conjugates of claim 2, wherein the contacting takesplace under conditions effective to allow hybridization of thecore/shell nanoparticle oligonucleotide conjugates with the boundnucleic acid; (b) subjecting the nanoparticle conjugate to an externalmagnetic field so as to accelerate movement of the nanoparticleconjugate to the surface to promote interaction between the nanoparticleconjugate and the nucleic acid; (c) removing from the surface anynanoparticle conjugates that have not hybridized with the nucleic acid;and (d) observing a detectable change brought about by hybridization ofthe nucleic acid with the nanoparticle conjugates.
 33. The method ofclaim 32 wherein the core/shell nanoparticle oligonucleotide conjugatecomprises Fe₃O₄/gold core/shell nanoparticles.
 34. The method of claim32 wherein step (c) is performed by rinsing the surface with a washsolution or reversing the magnetic field.
 35. A method of detecting atarget analyte bound to a surface comprising: (a) providing a surfacethat includes a bound target analyte; (b) contacting the surface with asolution comprising core/shell nanoparticle receptor conjugate, whereinthe receptor specifically binds to the analyte, wherein the contactingtakes place under conditions effective to allow binding of thenanoparticle conjugates with the bound nucleic acid; (c) subjecting thenanoparticle conjugate to an external magnetic field so as to acceleratemovement of the nanoparticle conjugate to the surface to promote bindinginteraction between the nanoparticle conjugate and the target analyte;(c) removing from the surface any nanoparticle conjugates that have notbound with the target analyte; and (d) observing a detectable changebrought about by binding interaction of the target analyte with thenanoparticle conjugates.